GPR55 is a rhodopsin-like (Class A) G protein-coupled receptor (GPCR), highly expressed in human striatum (Genbank accession # NM-005683). Characterizations of GPR55(-/-) (knock-out) mice reveal a role for the GPR55 receptor in inflammatory pain, neuropathic pain, and bone development; while other studies indicate that GPR55 activation is pro-carcinogenic. GPR55 may also be a cannabinoid receptor, since CB1/CB2 agonists, as well as antagonists have been reported to act at GPR55. Thus, GPR55 may also have a role to play in drug abuse. Despite these potential therapeutic uses, no low nanomolar potency ligands have been confirmed for this receptor, nor is there a radioligand developed to characterize binding at this receptor. The lack of such GPR55 ligands is a critical barrier to progress in this field. During a collaborative project between our individual laboratoris and the Sanford-Burnham screening center of the Molecular Libraries Probe Production Centers Network (MLPCN), we identified a series of GPR55 antagonists that belong to novel, unreported GPR55 antagonist chemotypes with IC50s in the 0.34 to 2.72 M range. The compounds that were in this potency range were also screened for agonist activity against GPR55 along with agonist/antagonist activity against GPR35, CB1, and CB2. Importantly, many of the GPR55 antagonists were completely selective, with no observed activity in the assays for related GPCRs for concentrations up to 20 M. We propose here to leverage these promising high-content screen results using state-of-the-art structure- based design and cheminformatics tools combined with a synthesis strategy to develop an SAR for selected scaffolds that leads to the identification of low nanomolar IC50 GPR55 antagonists with high receptor selectivity.